Nucleic acids have conventionally been thought of as primarily playing an informational role in biological processes. Through a method known as Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, it has become clear that nucleic acids have three dimensional structural diversity not unlike proteins. SELEX is a method for the in vitro synthesis and selection of nucleic acid molecules with highly specific binding to target molecules. The SELEX process was first described by Gold and Tuerk in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, now U.S. Pat. No. 5,475,096, and thereafter in U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled “Methods for Identifying Nucleic Acid Ligands”, now U.S. Pat. No. 5,270,163 (see also WO 91/19813). See also Tuerk et al, Science 249:505-10 (1990).
Nucleic acid ligands or aptamers are nonencoding single-stranded nucleic acid (DNA or RNA) that have the property of binding specifically to a desired target compound or molecule, and that have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising segments of randomized sequences, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids that have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
Nucleic acid ligands possess a number of features that can render them useful as therapeutic agents. They can be made as relatively small (e.g., 8 kDa to 15 kDa) synthetic compounds and can be selected to possess high affinity and specificity for target molecules (equilibrium dissociation constants ranging from, for example, 0.05-10 nM). Aptamers embody both the affinity properties of monoclonal antibodies and single chain antibodies (scFv's) and the manufacturing ease similar to that of a small peptide. Initial studies demonstrated the in vitro use of aptamers for studying protein function, and more recent studies have confirmed the utility of these compounds for studying in vivo protein function (Floege et al, Am J Pathol 154:169-179 (1999), Ostendorf et al, J Clin Invest 104:913-923, (1999)). In addition, animal studies to date have shown that aptamers and compounds of similar composition are well tolerated, exhibit low or no immunogenicity, and are thus suitable for repeated administration as therapeutic compounds (Floege et al, Am J Pathol 154:169-179 (1999), Ostendorf et al, J Clin Invest 104:913-923 (1999), Griffin et al, Blood 81:3271-3276 (1993), Hicke et al, J Clin Invest 106:923-928 (2000)).
As synthetic compounds, site specific modifications (insertions or deletions) can be made to aptamers to rationally alter their bioavailability and mode of clearance. For example, it has been found that 2′-fluoro pyrimidine-modified aptamers in the 10 kDa to 12 kDa size range have a short circulating half-life (˜10 minutes) following bolus intravenous administration but that simple chemical modification of the aptamer or conjugation of the aptamer to a high molecular weight inert carrier molecule (e.g., PEG) increases circulating half-life substantially (6-12 hours) (Willis et al, Bioconjug Chem 9:573-582 (1998), Tucker et al, J Chromatogr Biomed Sci Appl 732:203-212 (1999), Watson et al, Antisense Nucleic Acid Drug Dev 10:63-75 (2000)). Bioactive and nuclease resistant single-stranded nucleic acid ligands comprising L-nucleotides have been described (Williams et al, Proc. Natl. Acad. Sci. 94:11285 (1997); U.S. Pat. No. 5,780,221; Leva et al, Chem. Biol. 9:351 (2002)). These “L-aptamers” are reportedly stable under conditions in which aptamers comprising nucleotides of natural handedness (D-nucleotides) (that is, “D-aptamers”) are subject to degradation.
A number of third parties have applied for and secured patents covering the identification, manufacture and use of aptamers. As stated above, Larry Gold and Craig Tuerk are generally credited with first developing the SELEX method for isolating aptamers, and their method is described in a number of United States patents including those mentioned above and U.S. Pat. Nos. 5,670,637, 5,696,249, 5,843,653, 6,110,900, and 5,270,163, as well as other described in the Detailed Description of the Invention. Thomas Bruice et al. reported a process for producing aptamers in U.S. Pat. No. 5,686,242, which differs from the original SELEX process reported by Tuerk and Gold because it employs strictly random oligonucleotides during the screening sequence. The oligonucleotides screened in the '242 patent process lack the oligonucleotide primers that are present in oligonucleotides screened in the SELEX process.
Several patents to Gold et al. relate to aptamers themselves. For example, U.S. Pat. No. 6,114,120 relates to an aptamer that binds to a cell macromolecule. U.S. Pat. No. 5,670,637 relates to aptamers that bind to proteins. U.S. Pat. No. 5,696,249 relates to an aptamer produced by the SELEX process.
Other patents have issued that are directed to aptamers against specific biological targets, and to the methods for identifying these aptamers. U.S. Pat. Nos. 5,756,291 and 5,582,981 to O'Toole, for example, disclose a method for detecting thrombin using a labeled aptamer that comprises a defined six nucleotide sequence. U.S. Pat. Nos. 5,527,894 and 5,637,461 of Gold et al. relate to methods of identifying aptamers against the tat protein. Other patents that disclose aptamers directed against specific biological targets include U.S. Pat. No. 5,496,938 (HIV-reverse transcriptase), U.S. Pat. No. 5,476,766 (thrombin), U.S. Pat. No. 5,459,015 (fibroblast growth factor), U.S. Pat. No. 5,472,841 (neutrophil elastase), U.S. Pat. No. 5,849,479 (vascular endothelial growth factor), U.S. Pat. No. 5,726,017 (HIV GAG), U.S. Pat. No. 5,731,144 (TGFβ), U.S. Pat. No. 5,827,456 (chorionic gonadotropin hormone), U.S. Pat. No. 5,780,228 (lectins), U.S. Pat. No. 5,766,853 (selectins), U.S. Pat. No. 5,674,685 (platelet derived growth factor), U.S. Pat. No. 5,763,173 (DNA polymerases), U.S. Pat. No. 6,140,490 (complement system proteins), and U.S. Pat. No. 5,869,641 (CD4).
Sullenger, Rusconi, Kontos and White in WO 0226932 A2 describe RNA aptamers that bind to coagulation factors, E2F family transcription factors, Ang1, Ang2, and fragments or peptides thereof, transcription factors, autoimmune antibodies and cell surface receptors useful in the modulation of hemostasis and other biologic events. (See also Rusconi et al, Thrombosis and Haemostasis 83:841-848 (2000), White et al, J. Clin Invest 106:929-34 (2000), Ishizaki et al, Nat Med 2:1386-1389 (1996), and Lee et al, Nat Biotechnol 15:41-45 (1997)).
A number of patents have also issued that relate to specific uses of aptamers. For example, Bruice et al. in U.S. Pat. No. 6,022,691 describe the use of aptamers identified by a SELEX-like process to detect drugs and other molecules in biological fluids. Gold et al. in U.S. Pat. No. 5,843,653 provide a diagnostic method using aptamers. U.S. Pat. No. 6,110,900 discloses a diagnostic composition that contains an aptamer. U.S. Pat. No. 5,789,163 discloses a sandwich assay that employs aptamers as the capture and/or detection ligand. U.S. Pat. No. 6,147,204 describes the use of aptamers/lipophile complexes to deliver therapeutic and diagnostic aptamers to intracellular locations in vivo. U.S. Pat. Nos. 5,705,337, 5,962,219, 5,763,595 and 5,998,142 disclose aptamers that are chemically modified to covalently bind to target proteins.
Several methods have been developed that modify the base SELEX process to obtain aptamers that satisfy objectives in addition to exhibiting high binding affinity toward a target molecule. For example, a number of patents disclose the use of modified nucleotides in the SELEX process to obtain aptamers that exhibit improved properties. U.S. Pat. No. 5,660,985, for example, relates to SELEX using 2′-modified nucleotides that display enhanced in vivo stability. U.S. Pat. No. 6,083,696 discloses a “blended” SELEX process in which oligonucleotides covalently linked to non-nucleic acid functional units are screened for their capacity to bind a target molecule. Other patents describe post-SELEX modifications to aptamers to decrease their size, increase their stability, or increase target binding affinity. See, e.g., U.S. Pat. Nos. 5,817,785 and 5,648,214.
Still other patents describe unique SELEX processes. For example, U.S. Pat. Nos. 5,763,566 and 6,114,120 disclose processes for generating aptamers using the SELEX process with whole biological tissue as the target, to identify aptamers that have binding affinity toward biological tissues and components thereof U.S. Pat. No. 5,580,737 discloses a modification to the SELEX process that yields aptamers that can discriminate between two or more compounds. U.S. Pat. No. 5,567,588 discloses the “solution SELEX” method in which the nucleic acid candidate mixture is screened in solution in order to preferentially amplify the highest affinity aptamer.
Kauffman has obtained patents disclosing the generation of large libraries of proteins from large pools of stochastically generated oligonucleotide vectors. See U.S. Pat. Nos. 5,814,476 and 5,723,323.
Weis et al. disclose in U.S. Pat. No. 5,245,022 an oligonucleotide of about 12-25 bases that is terminally substituted by a polyalkyleneglycol. These modified oligonucleotides are reported to be resistant to exonuclease activity.
U.S. Pat. Nos. 5,670,633 and 6,005,087 to Cook et al. describe thermally stable 2′-fluoro oligonucleotides that are complementary to an RNA or DNA base sequence. U.S. Pat. Nos. 6,222,025 and 5,760,202 to Cook et al. describe the synthesis of 2′-O substituted pyrimidines and oligomers containing the modified pyrimidines. EP 0 593 901 B1 discloses oligonucleotide and ribozyme analogues with terminal 3′,3′- and 5′,5′-nucleoside bonds. U.S. Pat. No. 6,011,020 to Gold et at discloses an aptamer modified by polyethylene glycol.
A number of U.S. patents have issued that describe methods of large scale manufacturing that can be used to produce aptamers. Caruthers et al., for example, describe in U.S. Pat. Nos. 4,973,679; 4,668,777; and 4,415,732 a class of phosphoramidite compounds that are useful in the manufacture of oligonucleotides. In another series of patents, Caruthers et at disclose a method of synthesizing oligonucleotides using an inorganic polymer support. See, e.g., U.S. Pat. Nos. 4,500,707, 4,458,066 and 5,153,319. In still another series of patents, Caruthers et al. discloses a class of nucleoside phosphorodithioates that can be used to manufacture oligonucleotides. See, e.g., U.S. Pat. Nos. 5,278,302, 5,453,496 and 5,602,244. Reports of aptamers designed to bind to other aptamers include: Aldaz-Carroll L, Tallet B, Dausse E, Yurchenko L, Toulme J J.; Apical loop-internal loop interactions: a new RNA-RNA recognition motif identified through in vitro selection against RNA hairpins of the hepatitis C virus mRNA; Biochemistry. 2002 May 7;41(18):5883-93; Darfeuille F, Cazenave C, Gryaznov S, Duconge F, Di Primo C, Toulme J J.; RNA and N3′-→P5′ kissing aptamers targeted to the trans-activation responsive (TAR) RNA of the human immunodeficiency virus-1, Nucleosides Nucleotides Nucleic Acids. 2001 April-July ;20(4-7):441-9; Collin D, van Heijenoort C, Boiziau C, Toulme J J, Guittet E., NMR characterization of a kissing complex formed between the TAR RNA element of HIV-1 and a DNA aptamer. Nucleic Acids Res. 2000 Sep. 1;28(17):3386-91; Duconge F, Di Primo C, Toulme J J., Is a closing “GA pair” a rule for stable loop-loop RNA complexes? J Biol Chem. 2000 Jul. 14;275(28):21287-94.; Duconge F, Toulme J J. In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1. RNA. 1999Dc.;5(12):1605-14.; Boiziau C, Dausse E, Yurchenko L, Toulme J J., DNA aptamers selected against the HIV-1 trans-activation-responsive RNA element form RNA-DNA kissing complexes; J Biol Chem. 1999 Apr. 30;274(18): 12730-7; and Le Tinevez R, Mishra R K, Toulme J J., Selective inhibition of cell-free translation by oligonucleotides targeted to a mRNA hairpin structure; Nucleic Acids Res. 1998 May 15;26(10):2273-8.
Currently, many drugs elicit medical complications such as side effects and undesirable or uncontrollable outcomes. Treating medical complications that result from side effects leads to additional healthcare costs. The recent identification of this range of nucleic acid ligands useful in medical therapy has opened new avenues of research and development. While progress has been made in this area, a strong need remains to provide methods and compositions to improve the manner in which these ligands are used and to increase their efficacy, to better control the process of therapy, and to provide therapies that have decreased side effects over traditional therapeutic methods. The present invention provides compounds, compositions and methods to improve the process of using nucleic acid ligands in medical therapy. The approach provided by the present invention allows for more control over the therapeutic effect, pharmacokinetics and duration of activity of nucleic acid ligands.